PNG1, a yeast gene encoding a highly conserved peptide:N-glycanase.

It has been proposed that cytoplasmic peptide:N-glycanase (PNGase) may be involved in the proteasome-dependent quality control machinery used to degrade newly synthesized glycoproteins that do not correctly fold in the ER. However, a lack of information about the structure of the enzyme has limited our ability to obtain insight into its precise biological function. A PNGase-defective mutant (png1-1) was identified by screening a collection of mutagenized strains for the absence of PNGase activity in cell extracts. The PNG1 gene was mapped to the left arm of chromosome XVI by genetic approaches and its open reading frame was identified. PNG1 encodes a soluble protein that, when expressed in Escherichia coli, exhibited PNGase activity. PNG1 may be required for efficient proteasome-mediated degradation of a misfolded glycoprotein. Subcellular localization studies indicate that Png1p is present in the nucleus as well as the cytosol. Sequencing of expressed sequence tag clones revealed that Png1p is highly conserved in a wide variety of eukaryotes including mammals, suggesting that the enzyme has an important function.

Meiosis. DNA branching during meiotic recombination.

The branched forms of chromosomal DNA that arise during meiotic prophase in yeast have been characterized eletrophoretically, contributing to our understanding of meiotic synapsis and crossing over.

Meiotic induction of the yeast HOP1 gene is controlled by positive and negative regulatory sites.

The process of meiosis and sporulation in the yeast Saccharomyces cerevisiae is a highly regulated developmental pathway dependent on genetic as well as nutritional signals. The HOP1 gene, which encodes a component of meiotic chromosomes, is not expressed in mitotically growing cells, but its transcription is induced shortly after yeast cells enter the meiotic pathway. Through a series of deletions and mutations in the HOP1 promoter, we located two regulatory sites that are essential for proper regulation of HOP1. One site, called URS1H, brings about repression of HOP1 in mitotic cells and functions as an activator sequence in cells undergoing meiosis. The second site, which we designated UASH, acts as an activator sequence in meiotic cells and has similarity to the binding site of the mammalian CCAAT/enhancer binding protein (C/EBP). Both sites are required for full meiotic induction of the HOP1 promoter. We conclude that in mitotic yeast cells, the URS1H site maintains the repressed state of the HOP1 promoter, masking the effect of the UASH site. Upon entry into meiosis, repression is lifted, allowing the URS1H and UASH sites to activate high-level transcription.

DNA-binding activities of Hop1 protein, a synaptonemal complex component from Saccharomyces cerevisiae.

The meiosis-specific HOP1 gene is important both for crossing over between homologs and for production of viable spores. hop1 diploids fail to assemble synaptonemal complex (SC), which normally provides the framework for meiotic synapsis. Immunochemical methods have shown that the 70-kDa HOP1 product is a component of the SC. To assess its molecular function, we have purified Hop1 protein to homogeneity and shown that it forms dimers and higher oligomers in solution. Consistent with the zinc-finger motif in its sequence, the purified protein contained about 1 mol equivalent of zinc whereas mutant protein lacking a conserved cysteine within this motif did not. Electrophoretic gel mobility shift assays with different forms of M13 DNA showed that Hop1 binds more readily to linear duplex DNA and negatively superhelical DNA than to nicked circular duplex DNA and even more weakly to single-stranded DNA. Linear duplex DNA binding was enhanced by the addition of Zn2+, was stronger for longer DNA fragments, and was saturable to about 55 bp/protein monomer. Competitive inhibition of this binding by added oligonucleotides suggests preferential affinity for G-rich sequences and weaker binding to poly(dA-dT). Nuclear extracts of meiotic cells caused exonucleolytic degradation of linear duplex DNA if the extracts were prepared from hop1 mutants; addition of purified Hop1 conferred protection against this degradation. These findings suggest that Hop1 acts in meiotic synapsis by binding to sites of double-strand break formation and helping to mediate their processing in the pathway to meiotic recombination.

Meiotic segregation, synapsis, and recombination checkpoint functions require physical interaction between the chromosomal proteins Red1p and Hop1p.

In yeast, HOP1 and RED1 are required during meiosis for proper chromosome segregation and the consequent formation of viable spores. Mutations in either HOP1 or RED1 create unique as well as overlapping phenotypes, indicating that the two proteins act alone as well as in concert with each other. To understand which meiotic processes specifically require Red1p-Hop1p hetero-oligomers, a novel genetic screen was used to identify a single-point mutation of RED1, red1-K348E, that separates Hop1p binding from Red1p homo-oligomerization. The Red1-K348E protein is stable, phosphorylated in a manner equivalent to Red1p, and undergoes efficient homo-oligomerization; however, its ability to interact with Hop1p both by two-hybrid and coimmunoprecipitation assays is greatly reduced. Overexpression of HOP1 specifically suppresses red1-K348E, supporting the idea that the only defect in the protein is a reduced affinity for Hop1p. red1-K348E mutants exhibit reduced levels of crossing over and spore viability and fail to undergo chromosome synapsis, thereby implicating a role for Red1p-Hop1p hetero-oligomers in these processes. Furthermore, red1-K348E suppresses the sae2/com1 defects in meiotic progression and sporulation, indicating a previously unknown role for HOP1 in the meiotic recombination checkpoint.

A conditional allele of the Saccharomyces cerevisiae HOP1 gene is suppressed by overexpression of two other meiosis-specific genes: RED1 and REC104.

The HOP1 gene of Saccharomyces cerevisiae is believed to encode a protein component of the synaptonemal complex, the structure formed when homologous chromosomes synapse during meiotic prophase. Five new mutant alleles (three conditional, two nonconditional) of HOP1 were identified by screening EMS-mutagenized cells for a failure to complement the spore viability defect of a hop1 null allele. Two high copy plasmids were found that partially suppress the temperature-sensitive spore inviability phenotype of one of these alleles, hop1-628. The suppression is allele-specific; no effect of the plasmids is observed in hop1 null diploids. Mutation of either of the two suppressor genes results in recessive spore lethality, indicating that these genes play important roles during meiosis. The DNA sequence of one high copy suppressor gene matched that of RED1, a previously identified meiosis-specific gene. Our data strongly support the idea that RED1 protein is also a component of the synaptonemal complex and further suggest that the RED1 and HOP1 gene products may interact. The second suppressor maps to the right arm of chromosome VIII distal to CDC12 and is REC104, a meiosis-specific gene believed to act early in meiosis.

HOP1: a yeast meiotic pairing gene.

The recessive mutation, hop1-1, was isolated by use of a screen designed to detect mutations defective in homologous chromosomal pairing during meiosis in Saccharomyces cerevisiae. Mutants in HOP1 displayed decreased levels of meiotic crossing over and intragenic recombination between markers on homologous chromosomes. In contrast, assays of the hop1-1 mutation in a spo13-1 haploid disomic for chromosome III demonstrated that intrachromosomal recombination between directly duplicated sequences was unaffected. The spores produced by SPO13 diploids homozygous for hop1 were largely inviable, as expected for a defect in interhomolog recombination that results in high levels of nondisjunction. HOP1 was cloned by complementation of the spore lethality phenotype and the cloned gene was used to map HOP1 to the LYS11-HIS6 interval on the left arm of chromosome IX. Electron microscopy revealed that diploids homozygous for hop1 fail to form synaptonemal complex, which normally provides the structural basis for homolog pairing. We propose that HOP1 acts in meiosis primarily to promote chromosomal pairing, perhaps by encoding a component of the synaptonemal complex.

Genetic interactions between HOP1, RED1 and MEK1 suggest that MEK1 regulates assembly of axial element components during meiosis in the yeast Saccharomyces cerevisiae.

During meiosis, axial elements are generated by the condensation of sister chromatids along a protein core as precursors to the formation of the synaptonemal complex (SC). Functional axial elements are essential for wild-type levels of recombination and proper reductional segregation at meiosis I. Genetic and cytological data suggest that three meiosis-specific genes, HOP1, RED1 and MEK1, are involved in axial element formation in the yeast Saccharomyces cerevisiae. HOP1 and RED1 encode structural components of axial elements while MEK1 encodes a putative protein kinase. Using a partially functional allele of MEK1, new genetic interactions have been found between HOP1, RED1 and MEK1. Overexpression of HOP1 partially suppresses the spore inviability and recombination defects of mek1-974; in contrast, overexpression of RED1 exacerbates the mek1-974 spore inviability. Co-overexpression of HOP1 and RED1 in mek1-974 diploids alleviates the negative effect of overexpressing RED1 alone. Red1p/Red1p as well as Hop1p/Red1p interactions have been reconstituted in two hybrid experiments. Our results suggest a model whereby Mek1 kinase activity controls axial element assembly by regulating the affinity with which Hop1p and Red1p interact with each other.

Insertional mutations in the yeast HOP1 gene: evidence for multimeric assembly in meiosis.

The HOP1 gene of Saccharomyces cerevisiae has been shown to play an important role in meiotic synapsis. In this study we analyzed the mechanism of this function by phenotypic characterization of novel in-frame linker-insertion mutations located at various sites throughout the HOP1 coding sequence. Among 12 mutations found to cause defects in meiotic recombination and spore viability, three were temperature-sensitive for the spore viability defect. Although substantial meiotic recombination was found for these conditional alleles at the restrictive temperature, the level of exchange measured in spo13 meiosis was reduced in some of the monitored intervals, indicating that nondisjunction resulting from a deficit in crossing over could account for SPO13 spore inviability. Intragenic complementation between linker-insertion alleles was assessed by testing the viability of spores generated from heteroallelic diploids after SPO13 meiosis. Complex patterns of complementation and enhancement of the spore-inviability phenotype indicate that HOP1 functions in a multimeric complex. In addition, the ability of alleles which map near the carboxyl terminus to complement several other alleles provides evidence for a functional domain in this region of the protein. Two previously identified multicopy suppressors of the conditional hop1-628ts allele were tested for their effects in cells bearing the linker-insertion hop1 alleles. Overexpression of REC104 from a 2 mu plasmid was shown to enhance the spore viability of every allele tested, including a hop1 disruption allele. On the other hand, suppression by overexpression of RED1 from a 2 mu plasmid was found only for allele hop1-628ts. Surprisingly, similar overexpression of RED1 in strains bearing several other conditional hop1 linker-insertion alleles caused enhanced spore lethality. This finding, in conjunction with the evidence for a carboxy-terminal domain, provides new insight into the nature of interactions between the HOP1 and RED1 products in meiosis.

A role for MMS4 in the processing of recombination intermediates during meiosis in Saccharomyces cerevisiae.

The MMS4 gene of Saccharomyces cerevisiae was originally identified due to its sensitivity to MMS in vegetative cells. Subsequent studies have confirmed a role for MMS4 in DNA metabolism of vegetative cells. In addition, mms4 diploids were observed to sporulate poorly. This work demonstrates that the mms4 sporulation defect is due to triggering of the meiotic recombination checkpoint. Genetic, physical, and cytological analyses suggest that MMS4 functions after the single end invasion step of meiotic recombination. In spo13 diploids, red1, but not mek1, is epistatic to mms4 for sporulation and spore viability, suggesting that MMS4 may be required only when homologs are capable of undergoing synapsis. MMS4 and MUS81 are in the same epistasis group for spore viability, consistent with biochemical data that show that the two proteins function in a complex. In contrast, MMS4 functions independently of MSH5 in the production of viable spores. We propose that MMS4 is required for the processing of specific recombination intermediates during meiosis.

Bio-inspired CO2 conversion by iron sulfide catalysts under sustainable conditions.

The mineral greigite presents similar surface structures to the active sites found in many modern-day enzymes. We show that particles of greigite can reduce CO2 under ambient conditions into chemicals such as methanol, formic, acetic and pyruvic acid. Our results also lend support to the Origin of Life theory on alkaline hydrothermal vents.

A 64-channel transmitter for investigating parallel transmit MRI.

Multiple channel radiofrequency (RF) transmitters are being used in magnetic resonance imaging to investigate a number of active research topics, including transmit SENSE and B(1) shimming. Presently, the cost and availability of multiple channel transmitters restricts their use to relatively few sites. This paper describes the development and testing of a relatively inexpensive transmit system that can be easily duplicated by users with a reasonable level of RF hardware design experience. The system described here consists of 64 channels, each with 100 W peak output level. The hardware is modular at the level of four channels, easily accommodating larger or smaller channel counts. Unique aspects of the system include the use of vector modulators to replace more complex IQ direct digital modulators, 100 W MOSFET RF amplifiers with partial microstrip matching networks, and the use of digital potentiometers to replace more complex and costly digital-to-analog converters to control the amplitude and phase of each channel. Although mainly designed for B(1) shimming, the system is capable of dynamic modulation necessary for transmit SENSE by replacing the digital potentiometers controlling the vector modulators with commercially available analog output boards. The system design is discussed in detail and bench and imaging data are shown, demonstrating the ability to perform phase and amplitude control for B(1) shimming as well as dynamic modulation for transmitting complex RF pulses.

The HOP1 gene encodes a meiosis-specific component of yeast chromosomes.

The HOP1 gene in Saccharomyces cerevisiae is important for meiotic chromosomal pairing, because hop1 diploids fail to form synaptonemal complex during meiosis and are defective in crossing over between, but not within, chromosomes. We demonstrate here that the HOP1 gene is transcriptionally regulated during sporulation and that the HOP1 protein is situated along the lengths of meiotic chromosomes. Furthermore, the HOP1 protein contains a Cys2/Cys2 zinc finger motif. A mutation within this motif that changes a cysteine to serine results in the hop1 phenotype, consistent with the possibility that the HOP1 gene product acts in chromosome synapsis by directly interacting with DNA. These observations demonstrate that HOP1 encodes a component of meiotic chromosomes, perhaps serving as a constituent of the synaptonemal complex.

Conserved properties between functionally distinct MutS homologs in yeast.

In the yeast Saccharomyces cerevisiae there are five nuclear MutS homologs that act in two distinct processes. MSH2, 3, and 6 function in mismatch repair in both vegetative and meiotic cells, whereas MSH4 and MSH5 act specifically to facilitate crossovers between homologs during meiosis. Coimmunoprecipitation as well as two-hybrid experiments indicate that the Msh4 and Msh5 proteins form a hetero-oligomeric structure similar to what is observed for the Msh proteins involved in mismatch repair. Mutation of conserved amino acids in the NTP binding and putative helix-turn-helix domains of Msh5p abolish function but are still capable of interaction with Msh4p, suggesting that NTP binding plays a role downstream of hetero-oligomer formation. No hetero-oligomers are observed between the mismatch repair MutS proteins (Msh2p and Msh6p) and either Msh4p or Msh5p. These results indicate that one level of functional specificity between the mismatch repair and meiotic crossover MutS homologs in yeast is provided by the ability to form distinct hetero-oligomers.

MSH5, a novel MutS homolog, facilitates meiotic reciprocal recombination between homologs in Saccharomyces cerevisiae but not mismatch repair.

Using a screen designed to identify yeast mutants specifically defective in recombination between homologous chromosomes during meiosis, we have obtained new alleles of the meiosis-specific genes, HOP1, RED1, and MEK1. In addition, the screen identified a novel gene designated MSH5 (MutS Homolog 5). Although Msh5p exhibits strong homology to the MutS family of proteins, it is not involved in DNA mismatch repair. Diploids lacking the MSH5 gene display decreased levels of spore viability, increased levels of meiosis I chromosome nondisjuction, and decreased levels of reciprocal exchange between, but not within, homologs. Gene conversion is not reduced. Msh5 mutants are phenotypically similar to mutants in the meiosis-specific gene MSH4 (Ross-Macdonald and Roeder 1994). Double mutant analysis using msh4 msh5 diploids demonstrates that the two genes are in the same epistasis group and therefore are likely to function in a similar process--namely, the facilitation of interhomolog crossovers during meiosis.

Red1p, a MEK1-dependent phosphoprotein that physically interacts with Hop1p during meiosis in yeast.

The synaptonemal complex (SC) is a proteinaceous structure formed between pairs of homologous chromosomes during prophase I of meiosis. The proper assembly of axial elements (AEs), lateral components of the SC, during meiosis in the yeast, Saccharomyces cerevisiae, is essential for wild-type levels of recombination and for the accurate segregation of chromosomes at the first meiotic division. Genetic experiments have indicated that the stoichiometry between two meiosis-specific components of AEs in S. cerevisiae, HOP1 and RED1, is critical for proper assembly and function of the SC. A third meiosis-specific gene, MEK1, which encodes a putative serine/threonine protein kinase, is also important for proper AE function, suggesting that AE formation is regulated by phosphorylation. In this paper, we demonstrate that Mek1p is a functional kinase in vitro and that catalytic activity is an essential part of the meiotic function of Mek1 in vivo. Immunoblot analysis revealed that Red1p is a MEK1-dependent phosphoprotein. Co-immunoprecipitation experiments demonstrated that the interaction between Hop1p and Red1p is enhanced by the presence of MEK1. Thus, MEK1-dependent phosphorylation of Red1p facilitates the formation of Hop1p/Red1p hetero-oligomers, thereby enabling the formation of functional AEs.